1. Field of the Invention
The present invention describes the DNA sequence for eukaryotic genes encoding xcex5 cyclase, isopentenyl pyrophosphate isomerase (IPP) and xcex2-carotene hydroxylase as well as vectors containing the same and hosts transformed with said vectors. The present invention also provides a method for augmenting the accumulation of carotenoids and production of novel and rare carotenoids. The present invention provides methods for controlling the ratio of various carotenoids in a host. Additionally, the present invention provides a method for screening for eukaryotic genes encoding enzymes of carotenoid biosynthesis and metabolism.
2. Discussion of the Background
Carotenoid pigments with cyclic endgroups are essential components of the photosynthetic apparatus in oxygenic photosynthetic organisms (e.g., cyanobacteria, algae and plants; Goodwin, 1980). The symmetrical bicyclic yellow carotenoid pigment xcex2-carotene (or, in rare cases, the asymmetrical bicyclic xcex1-carotene) is intimately associated with the photosynthetic reaction centers and plays a vital role in protecting against potentially lethal photooxidative damage (Koyama, 1991). xcex2-carotene and other carotenoids derived from it or from xcex1-carotene also serve as light-harvesting pigments (Siefermann-Harms, 1987), are involved in the thermal dissipation of excess light energy captured by the light-harvesting antenna (Demmig-Adams and Adams, 1992), provide substrate for the biosynthesis of the plant growth regulator abscisic acid (Rock and Zeevaart, 1991; Parry and Horgan, 1992), and are precursors of vitamin A in human and animal diets (Krinsky, 1987). Plants also exploit carotenoids as coloring agents in flowers and fruits to attract pollinators and agents of seed dispersal (Goodwin, 1980). The color provided by carotenoids is also of agronomic value in a number of important crops. Carotenoids are currently harvested from plants for use as pigments in food and feed.
The probable pathway for formation of cyclic carotenoids in plants, algae and cyanobacteria is illustrated in FIG. 1. Two types of cyclic endgroups are commonly found in higher plant carotenoids, these are referred to as the xcex2 and xcex5 cyclic endgroups (FIG. 3.; the acyclic endgroup is referred to as the xcexa8 or psi endgroup). These cyclic endgroups differ only in the position of the double bond in the ring. Carotenoids with two xcex2 rings are ubiquitous, and those with one xcex2 and one xcex5 ring are common, but carotenoids with two xcex5 rings are rarely detected. xcex2-Carotene (FIG. 1) has two xcex2 endgroups and is a symmetrical compound that is the precursor of a number of other important plant carotenoids such as zeaxanthin and violaxanthin (FIG. 2).
Carotenoid enzymes have previously been isolated from a variety of sources including bacteria (Armstrong et al., 1989, Mol. Gen. Genet. 216, 254-268; Misawa et al., 1990, J. Bacteriol., 172, 6704-12), fungi (Schmidhauser et al., 1990, Mol. Cell. Biol. 10, 5064-70), cyanobacteria (Chamovitz et al., 1990, Z. Naturforsch, 45c, 482-86) and higher plants (Bartley et al., Proc. Natl. Acad. Sci USA 88, 6532-36; Martinez-Ferez and Vioque, 1992, Plant Mol. Biol. 18, 981-83). Many of the isolated enzymes show a great diversity in function and inhibitory properties between sources. For example, phytoene desaturases from Synechococcus and higher plants carry out a two-step desaturation to yield xcex6-carotene as a reaction product; whereas the same enzyme from Erwinia introduces four double bonds forming lycopene. Similarity of the amino acid sequences are very low for bacterial versus plant enzymes. Therefore, even with a gene in hand from one source, it is difficult to screen for a gene with similar function in another source. In particular, the sequence similarity between prokaryotic and eukaryotic genes is quite low.
Further, the mechanism of gene expression in prokaryotes and eukaryotes appears to differ sufficiently such that one can not expect that an isolated eukaryotic gene will be properly expressed in a prokaryotic host.
The difficulties in isolating related genes is exemplified by recent efforts to isolated the enzyme which catalyzes the formation of xcex2-carotene from the acyclic precursor lycopene. Although this enzyme had been isolated in a prokaryote, it had not been isolated from any photosynthetic organism nor had the corresponding genes been identified and sequenced or the cofactor requirements established. The isolation and characterization of the enzyme catalyzing formation of xcex2-carotene in the cyanobacterium Synechococcus PCC7942 was described by the present inventors and others (Cunningham et al., 1993 and 1994).
The need remains for the isolation of eukaryotic genes involved in the carotenoid biosynthetic pathway, including a gene encoding an xcex5 cyclase, IPP isomerase and xcex2-carotene hydroxylase. There remains a need for methods to enhance the production of carotenoids. There also remains a need in the art for methods for screening for eukaryotic genes encoding enzymes of carotenoid biosynthesis and metabolism.
Accordingly, a first object of this invention is to provide isolated eukaryotic genes which encode enzymes involved in carotenoid biosynthesis; in particular, xcex5 cyclase, IPP isomerase and xcex2-carotene hydroxylase.
A second object of this invention is to provide eukaryotic genes which encode enzymes which produce novel carotenoids.
A third object of the present invention is to provide vectors containing said genes.
A fourth object of the present invention is to provide hosts transformed with said vectors.
Another object of the present invention is to provide hosts which accumulates novel or rare carotenoids or which overexpress known carotenoids.
Another object of the present invention is to provide hosts with inhibited carotenoid production.
Another object of this invention is to secure the expression of eukaryotic carotenoid-related genes in a recombinant prokaryotic host.
A final object of the present invention is to provide a method for screening for eukaryotic genes which encode enzymes involved in carotenoid biosynthesis and metabolism.
These and other objects of the present invention have been realized by the present inventors as described below.